Reversible nuclear genetic system for male sterility in transgenic plants

ABSTRACT

Plant development can be altered by transforming a plant with a genetic construct that includes regulatory elements and DNA sequences capable of acting in a fashion to inhibit pollen formation or function, thus rendering the transformed plant reversibly male-sterile. In particular, the present invention relates to the use of dominant negative genes and an anther-specific promoter. Male sterility is reversed by incorporation into a plant of a second genetic construct which represses the dominant negative gene. The invention also relates to novel DNA sequences which exhibit the ability to serve as anther-specific promoters in plants.

This application is a continuation of U.S. patent application Ser. No.08/470,354, filed Jun. 7, 1995, now U.S. Pat. No. 5,792,853, which is acontinuation-in-part of U.S. patent application Ser. No. 08/351,899,filed Dec. 8, 1994, now U.S. Pat. No. 5,750,868.

BACKGROUND OF THE INVENTION

Plant development can be altered, according to the present invention, bytransforming a plant with a genetic construct that includes regulatoryelements and structural genes capable of acting in a cascading fashionto produce a reversible effect on a plant phenotype. A suitableconstruct includes a tissue specific promoter, a dominant negative gene,and a nucleotide sequence encoding a transcriptional activator linked toa DNA binding protein. In particular, the present invention relates tothe use of a DAM-methylase gene as a dominant negative gene and ananther-specific promoter to produce transgenic plants that arereversibly male-sterile.

There is a need for a reversible genetic system for producing malesterile plants, in particular for autogamous plants. Production ofhybrid seed for commercial sale is a large and important industry.Hybrid plants grown from hybrid seed benefit from the heterotic effectsof crossing two genetically distinct breeding lines. The commerciallydesirable agronomic performance of hybrid offspring is superior to bothparents, typically in vigor, yield and uniformity. The betterperformance of hybrid seed varieties compared to open-pollinatedvarieties makes the hybrid seed more attractive for farmers to plant andtherefore commands a premium price in the market.

In order to produce hybrid seed uncontaminated with self-seed,pollination control methods must be implemented to ensurecross-pollination and to guard against self-pollination. Pollinationcontrol mechanisms include mechanical, chemical and genetic means.

A mechanical means for hybrid seed production can be used if the plantof interest has spatially separate male and female flowers or separatemale and female plants. For example, a maize plant has pollen-producingmale flowers in an inflorescence at the apex of the plant, and femaleflowers in the axiles of leaves along the stem. Outcrossing of maize isassured by mechanically detasseling the female parent to preventselfing. Even though detasseling is currently used in hybrid seedproduction for plants such as maize, the process is labor-intensive andcostly, both in terms of the actual detasseling cost and yield loss as aresult of detasseling the female parent.

Most major crop plants of interest, however, have both functional maleand female organs within the same flower, therefore, emasculation is nota simple procedure. While it is possible to remove by hand the pollenforming organs before pollen is shed, this form of hybrid production isextremely labor intensive and expensive. Seed is produced in this manneronly if the value and amount of seed recovered warrants the effort.

A second general means of producing hybrid seed is to use chemicals thatkill or block viable pollen formation. These chemicals, termedgametocides, are used to impart a transitory male-sterility. Commercialproduction of hybrid seed by use of gametocides is limited by theexpense and availability of the chemicals and the reliability and lengthof action of the applications. A serious limitation of gametocides isthat they have phytotoxic effects, the severity of which are dependenton genotype. Other limitations include that these chemicals may not beeffective for crops with an extended flowering period because newflowers produced may not be affected. Consequently, repeated applicationof chemicals is required.

Many current commercial hybrid seed production systems for field cropsrely on a genetic means of pollination control. Plants that are used asfemales either fail to make pollen, fail to shed pollen, or producepollen that is biochemically unable to effect self-fertilization. Plantsthat are unable to self-fertilize are said to be “self-incompatible”(SI). Difficulties associated with the use of a self-incompatibilitysystem include availability and propagation of the self-incompatiblefemale line, and stability of the self-compatibility. In some instances,self-incompatibility can be overcome chemically, or immature buds can bepollinated by hand before the bio-chemical mechanism that blocks pollenis activated. Self-incompatible systems that can be deactivated areoften very vulnerable to stressful climatic conditions that break orreduce the effectiveness of the biochemical block to self-pollination.

Of more widespread interest for commercial seed production are systemsof pollen-control-based genetic mechanisms causing male sterility. Thesesystems are of two general types: (a) genic male sterility, which is thefailure of pollen formation because of one or more nuclear genes or (b)cytoplasmic-genetic male sterility, commonly referred to as “cytoplasmicmale sterility” (CMS), in which pollen formation is blocked or abortedbecause of an alteration in a cytoplasmic organelle, which generally isa mitochondria.

Although there are hybridization schemes involving the use of CMS, thereare limitations to its commercial value. An example of a CMS system, isa specific mutation in the cytoplasmically located mitochondria whichcan, when in the proper nuclear background, lead to the failure ofmature pollen formation. In some instances, the nuclear background cancompensate for the cytoplasmic mutation and normal pollen formationoccurs. Specific nuclear “restorer genes” allow pollen formation inplants with CMS mitochondria. Generally, the use of CMS for commercialseed production involves the use of three breeding lines: a male-sterileline (female parent), a maintainer line which is isogeneic to themale-sterile line but contains fully functional mitochondria, and a maleparent line. The male parent line may carry the specific restorer genesand, hence, is usually designated a “restorer line,” which impartsfertility to the hybrid seed.

For crops such as vegetable crops for which seed recovery from thehybrid is unimportant, a CMS system can be used without restoration. Forcrops for which the fruit or seed of the hybrid is the commercialproduct, the fertility of the hybrid seed must be restored by specificrestorer genes in the male parent or the male-sterile hybrid must bepollinated. Pollination of non-restored hybrids can be achieved byincluding with hybrids a small percentage of male fertile plants toeffect pollination. In most species, the CMS trait is inheritedmaternally, since all cytoplasmic organelles are inherited from the eggcell only, and this restricts the use of the system.

CMS systems possess limitations that preclude them as a sole solution toproduction of male sterile plants. For example, one particular CMS typein maize (T-cytoplasm) confers sensitivity to the toxin produced byinfection by a particular fungus. Although still used for a number ofcrops, CMS systems may break down under certain environmentalconditions.

Nuclear (genic) sterility can be either dominant or recessive. Dominantsterility can only be used for hybrid seed formation if propagation ofthe female line is possible (for example, via in vitro clonalpropagation). Recessive sterility can be used if sterile and fertileplants are easily discriminated. Commercial utility of genic sterilitysystems is limited however by the expense of clonal propagation androguing the female rows of self-fertile plants.

Discovery of genes which would alter plant development would beparticularly useful in developing genetic methods to induce malesterility because other currently available methods, includingdetasseling, CMS and SI, have shortcomings.

A search for methods of altering development in plants by use of geneticmethods led to methylase genes of the present invention. Changes in theDNA methylation pattern of specific genes or promoters have accountedfor changes in gene expression. Methylation of DNA is a factor inregulation of genes during development of both plants and animals.

Methylation patterns are established by methods such as the use ofmethyl-sensitive CpG-containing promoters (genes). In general, activelytranscribed sequences are under methylated. In animals, sites ofmethylation are modified at CpG sites (residues). Genetic control ofmethylation of adenine (A) and cytosine (C) (nucleotides present in DNA)is affected by genes in bacterial and mammalian species. In plants,however, methyl moieties exist in the sequence CXG, where X can be A, Cor T, where C is the methylated residue. Inactivation due to methylationof A is not known in plants, particularly within GATC sites known to bemethylated in other systems.

Although there is no suggestion in the art that methylation might beinduced in tissues specifically or otherwise, to achieve a desired endin a transgenic plant, it was known in the art that promoter methylationcan cause gene inactivation, and alter the phenotype in transgenicorganisms.

Envisioning directed methylation as a means for control of plantdevelopment, for example, to effect male sterility, would be discouragedby difficulties anticipated in using expression of a gene that has ageneralized inactivating effect in a ubiquitous target, e.g., amethylase gene such as the E. coli DNA adenine methylase (DAM) for whichGATC is a target, as a means to control a specific developmental stepwithout otherwise deleteriously affecting the plant. The DAM targetexists in many promoters, therefore, a problem of maintaining plantviability would be expected from inactivating promoters and/or genesthat are crucial for cell viability. Unless there was a way to“compartmentalize” methylation introduced into a host system by anexogenous vector, methylation as an approach to producing male sterilityby genetic means would not be expected to succeed. The present inventionprovides methods and compositions to compartmentalize and to manipulategenes such as DAM to effect changes in plant development.

SUMMARY OF THE INVENTION

The invention relates to an isolated DNA molecule comprising anucleotide sequence of capable of regulating the expression of a DNAsequence in anther tissue when the DNA molecule is part of a recombinantDNA construct.

The isolated molecule may comprise the nucleotide sequence of theSca-NcoI fragment of DP5055, a nucleotide sequence extending at least503 base pairs upstream relative to the start codon at nucleotideposition 1488 of FIG. 1, a nucleotide sequence extending from position−503 to position −1 upstream relative to the start codon at nucleotideposition 1488 of FIG. 1, a nucleotide sequence extending from position−587 to position −1 upstream relative to the start codon at nucleotideposition 1488 of FIG. 1, a nucleotide sequence extending from position−890 to position −1 upstream relative to the start codon at nucleotideposition 1488 of FIG. 1, or a nucleotide sequence extending fromposition −503 to position −134 upstream relative to the start codon atnucleotide position 1488 of FIG. 1.

The invention further relates to a recombinant DNA construct comprising:a DNA sequence that encodes a gene product which, when expressed,inhibits pollen formation or function; an operator capable ofcontrolling the expression of the DNA sequence; a gene encoding a DNAbinding protein capable of binding to the operator and activatingtranscription of said dominant negative gene; and a tissue specificpromoter operably linked to DNA sequence.

The recombinant DNA construct of the invention may also comprise: a DNAsequence encoding a gene product which when expressed in a plantinhibits pollen formation or function; an operator which controls theexpression of said DNA sequence; and a promoter specific to cellscritical to pollen formation or function operatively linked to said DNAsequence encoding a gene product. In further embodiments, therecombinant DNA construct may further comprise a selectable marker gene,a DNA sequence encoding a DNA binding region, or a DNA sequence encodingan activating domain.

In one embodiment, the gene product encoded by the DNA sequence of therecombinant DNA construct of the invention may be a cytotoxin. Inanother embodiment, the promoter may be an anther-specific promoter, andconstruct may comprise the constructs DP5814, DP6509, PHP8036, PHP8037,or PHP6520. In still another embodiment, the operator may be lexAoperator. And, in yet another embodiment, the recombinant DNA constructmay further comprise a selectable marker gene.

In another embodiment of the invention, the recombinant DNA constructcomprises a DNA sequence encoding a DNA-binding protein, capable ofbinding to the operator of the recombinant DNA construct as definedabove, and a promoter which controls expression of said DNA sequence.This recombinant DNA construct may further comprise a selectable markergene. In one embodiment, the DNA binding protein of the recombinant DNAconstruct may be lexA protein. In another embodiment, the promoter maybe specific to cells critical to pollen formation or function. In stillanother embodiment, the promoter may be an anther specific promoter,which may comprise the isolated DNA molecule as defined above. Stillfurther, the promoter of this construct may be an inducible promoter ora constitutive promoters which may be maize ubiquitin promoter as theconstitutive promoter. The recombinant DNA construct may be PHP6522 orPHP6555.

An additional aspect of the invention relates to is an expression vectorcomprising the isolated DNA molecule as defined above. The expressionvector may further comprise a DNA sequence encoding a gene product, inwhich the DNA sequence is operably linked to the promoter. In oneembodiment, the gene product of the expression vector disrupts thefunction or formation of pollen. In still another embodiment, the DNAsequence of the expression vector is heterologous with respect to thepromoter. The invention also relates to a transgenic plant comprisingthe expression vector.

A further embodiment of the invention includes an anther specificpromoter comprising a nucleotide sequence of promoter 5126f whichexhibits the ability to control expression of a DNA sequence encoding agene product. In one embodiment of the invention the gene productinhabits the function or formation of pollen. In another embodiment, thegene product comprises a cytoxin.

Yet another aspect of the invention relates to a method for producingreversible male sterility in plants. The method comprises the steps (a)transforming a first plant with an recombinant DNA construct such thatthe plant exhibits male sterility, the construct comprising (i) a lexAoperator controlling the expression of a DNA sequence that encodes agene product which inhibits the function or formation of pollen, theoperator embedded in a tissue specific promoter which is operativelylinked to the DNA sequence, and (ii) a DNA sequence encoding a lexArepressor, the DNA sequence operatively linked to an inducible promoter;and (b) exposing the plant to an inducer to reverse the male sterileeffect of the construct. In further embodiments, the tissue specificpromoter may be an anther-specific promoter. In another embodiment ofthe invention, the anther-specific promoter may comprise a nucleotidesequence of promoter 5126 which exhibits the ability to controlexpression of a DNA sequence encoding a gene product. In yet anotherembodiment the gene product may be a dominant negative gene, which maybe DAM-methylase.

Also, the present invention relates to a male sterile plant and a methodof producing a male sterile plant which comprises: (a) introducing intothe genome of a pollen producing plant capable of being geneticallytransformed a recombinant DNA molecule comprising (i) a DNA sequenceencoding a gene product which when expressed in a plant inhibits pollenformation or function, (ii) an operator which controls the expression ofthe DNA sequence, and (iii) a promoter specific to cells critical topollen formation or function operatively linked to the DNA sequenceencoding a gene product; and (b) growing said pollen-producing plantunder conditions such that male sterility is achieved as a result of theexpression of the DNA sequence. In further embodiments of this aspect ofthe invention the gene product may be a cytotoxin. In still anotherembodiment, the promoter of the invention may be an anther-specificpromoter. In yet another embodiment, the anther-specific promoter maycomprise a nucleotide sequence of promoter 5126 which exhibits theability to control expression of a DNA sequence encoding a gene product.In yet another embodiment, the operator may be lexA operator. The methodof producing a male sterile plant may further comprise a selectablemarker gene.

The invention further relates to hybrid seed and a method of producinghybrid seed from a male sterile plant which comprises (a) introducinginto the genome of a pollen producing plant capable of being geneticallytransformed a recombinant DNA molecule comprising (i) a DNA sequenceencoding a gene product which when expressed in a plant inhibits pollenformation or function, (ii) an operator which controls the expression ofthe DNA sequence, and (iii) a promoter specific to cells critical topollen formation or function operatively linked to the DNA sequenceencoding a gene product; (b) growing the pollen-producing plant underconditions such that male sterility is achieved as a result of theexpression of the DNA sequences; (c) crossing the male sterile plantwith pollen derived from a male fertile line, the pollen havingintegrated into its genome a recombinant DNA molecule comprising a DNAsequence encoding a DNA-binding protein and a promoter which controlsexpression of the DNA sequence, the protein capable of binding to theoperator of the recombinant DNA of the male-sterile plant; and (d)harvesting the hybrid seed with restored fertility. In a furtherembodiment of this aspects of the invention, the gene product may becytotoxin. In still another embodiment, the promoter may be ananther-specific promoter. In still another embodiment of the invention,the anther-specific promoter may comprise a nucleotide sequence ofpromoter 5126 which exhibits the ability to control expression of a DNAsequence encoding a gene product. In yet another embodiment, theoperator may be lexA operator. The method of producing a male sterileplant may further comprise a selectable marker gene.

Also an aspect of the invention is a method of producing reversible malesterility in a plant which comprises: (a) introducing into the genome ofa pollen producing plant capable of being genetically transformed afirst recombinant DNA molecule comprising (i) a DNA sequence encoding agene product which when expressed in a plant inhibits pollen formationor function, (ii) an operator which controls the expression of the DNAsequence, and (iii) a promoter specific to cells critical to pollenformation or function operatively linked to the DNA sequence encoding agene product; (b) growing the pollen-producing plant under conditionssuch that male sterility is achieved as a result of the expression ofthe DNA sequences; and (c) crossing the male sterile plant with pollenderived from a male fertile line to form a hybrid plant which is malefertile, the pollen having integrated into its genome a secondrecombinant DNA molecule comprising a DNA sequence encoding aDNA-binding protein and a promoter which controls expression of the DNAsequence, the protein capable of binding to the operator of therecombinant DNA of the male-sterile plant. In further embodiments ofthis aspect of the invention the gene product may be cytotoxin. In stillanother embodiment, the promoter may be an anther-specific promoter. Inyet another embodiment of the invention, the anther-specific promotermay comprise a nucleotide sequence of promoter 5126 which exhibits theability to control expression of a DNA sequence encoding a gene product.In yet another embodiment, the operator may be lexA operator. In oneembodiment, the first recombinant molecule or second recombinant DNAmolecule may further comprises a selectable marker gene. In anotherembodiment of the invention, the DNA-binding protein may be lexAprotein. In yet another embodiment, the promoter of the secondrecombinant DNA molecule is a promoter specific to cells critical topollen formation or function, and may be an anther-specific promoter.The anther-specific promoter may comprise an isolated DNA moleculecomprising a nucleotide sequence of capable of regulating the expressionof a DNA sequence in anther tissue when the DNA molecule is part of anoperable recombinant DNA construct. The promoter of the secondrecombinant DNA molecule may be an inducible promoter or a constitutivepromoter, which may be maize ubiquitin promoter.

Another aspect of the present invention is a transformed plant cell, anda plant regenerated from such plant cell, containing an expressionvector comprising an isolated DNA molecule comprising a nucleotidesequence of capable of regulating the expression of a DNA sequence inanther tissue when the DNA molecule is part of an operable recombinantDNA construct. The expression vector may further comprise a DNA sequenceencoding a gene product, the sequence being operable linked to thepromoter. The invention also relates to hybrid seed and make sterileplants produced by the methods of the invention.

In accordance with the present invention, two types of genetic systemshave been combined in a transforming genetic construct to create acascading mechanism to affect plant development. One system highlights atissue-specific promoter which controls gene expression, e.g.,expression of a transcriptional activator. The second system includes aDNA sequence that encodes a gene product which inhibits pollen formationor function, e.g., a dominant negative gene such as a methylase gene,the expression product of which disrupts pollen formation and function.

A specific component of the invention is a transforming geneticconstruct, incorporating elements of both of these systems, thatincludes regulatory elements and structural genes capable of interactingto cause a particular phenotype, depending on the specific regulatorsand genes present. By virtue of the presence of this construct in oneparent plant, certain advantages of the present invention arise. Forexample, a one-step approach to achieving male sterility is implemented.For example, the present invention contemplates the use, in producingreversible male sterility in plants, of a genetic construct thatcontains a tissue-specific promoter, a dominant negative gene, and aspecific stretch of DNA that encloses a transcriptional activator whichis capable of activating the dominant negative gene. The presentinvention in one aspect thus provides a new, nuclear basis formanipulating male fertility.

More specifically, a genetic construct suitable for the presentinvention comprises a dominant negative gene and a specific stretch ofDNA that, when positioned upstream of the dominant negative gene,controls expression of the dominant negative gene in association with aDNA binding gene and a promoter that controls expression at a specifictime or times in development.

A dominant negative gene is one that, when expressed, effects a dominantphenotype in the plant. Herskowitz (1987), used the term “dominantnegative” to denote a gene that encodes a mutant polypeptide which, whenover-expressed, disrupts the activity of the wild-type gene. A wild typegene is one from which the mutant derived. In the present descriptionthe phrase “dominant negative gene” is applied to a gene coding for aproduct that disrupts an endogenous genetic process of a host cell whichreceives the gene, and that is effective in a single copy or may producean effect due to overexpression of the gene either by increasedproduction of the gene product, or by coexpression of multiple copies ofthe gene. Exemplary of the class of dominant negative genes arecytotoxic genes, methylase genes, and growth-inhibiting genes. Dominantnegative genes include diphtheria toxin A-chain gene (Czako and An,1991), cell cycle division mutants such as CDC in maize (Colasanti, etal., 1991) the WT gene (Farmer, et al., 1994) and P68 (Chen, et al.,1991). Candidate genes for a dominant negative gene in the geneticconstructs of the present invention are also exemplified by aDAM-methylase gene, such as the gene isolated from E. coli. A candidategene may or may not be deleterious to the source from which it wasderived. Indeed, a candidate gene may serve an essential function in itssource.

In an illustrative embodiment, a candidate dominant negative gene whichexploits genetic methylation to alter development of specific planttissues is a DAM-methylase gene. This gene is used to inactivate agenetic region critical for pollen formation or function thereby causinga male sterile plant to form.

In particular, the components of a first genetic construct of thepresent invention are as follows:

A transcriptional activator, such as the maize C1 gene, is fused to abacterial DNA binding protein such as lexA. (Brent and Ptashne, 1985).This gene fusion, designated “lexA-C1,” is placed under the control ofan anther-specific promoter, such as the 5126 promoter. The geneticconstruct is designated as:

5126::lexA-C1

The DAM-methylase gene is placed behind a minimal 35S promotercontaining the lexA binding site (Lex), as symbolized below:

35S-lexAop::DAM

35S-lexAop::DAM and 5126::lexA-C1 are two separate transcription unitson the same plasmid, the plasmid preferably including a selectablemarker gene.

A transgenic plant containing a construct of the present invention canbe regenerated from a culture transformed with that same construct, solong as plant species involved is susceptible to regeneration.

A plant is regenerated from a transformed cell or culture, or from anexplant, by methods disclosed herein and known to those of skill in theart. “Culture” in this context comprehends an aggregate of cells, acallus, or derivatives thereof that are suitable for culture. Methodsvary according to the plant species. Seed is obtained from theregenerated plant or from a cross between the regenerated plant and asuitable plant of the same species using breeding methods known to thoseof skill in the art.

When a first construct, as that described above, is transformed intoplants, the result is increased expression compared to the situationwhere transcription is controlled only by the anther-specific promoterof the DAM-methylase gene. The enhanced expression is due to productionof the transcriptional activator lexA-C1, which specifically binds tothe Lex operator and controls the expression of the DAM-methylase gene,effecting male-sterility. The methods of the present invention areparticularly attractive for expression of genes, such as those in maize,that when mutated confer a dominant negative phenotype. Gene productsencoded by such genes generally require high expression in order tointerfere with the function of the wild-type protein, e.g., the maizeCDC21 gene.

To reverse this effect, a first plant having the first construct ismated with a second plant that contains a second construct including the5126 or other suitable promoter, including other anther-specificpromoters such as the 5126 deletion mutation promoters or constitutivepromoters, fused to the lexA gene which expresses only the DNA bindingprotein lexA. This protein binds specifically to the LexA operator butdoes not activate gene expression. Rather, it represses expression, thusshutting off DAM-methylase gene expression and rendering a plant havingboth a first and a second genetic construct, male-fertile.

Pursuant to the present invention, another way to utilize the componentsof this system is to embed a lexA DNA binding site (i.e., lexA operator)in the tissue specific promoter 5126 and couple the expression of thelexA repressor to an inducible promoter. Any gene that is expressed dueto transcription of the 5126 promoter is turned off (repressed) byapplying a chemical which induces the expression of lexA. LexA repressorprotein binds to the lexAop located in the 5126 promoter and, as aconsequence of binding to this region of DNA, shuts off expression ofthe reporter gene. If, for example, this system is used with the DAMmethylase gene, application of a chemical inducer reverses the sterilephenotype and renders the plant male-fertile.

By way of example, a suitable genetic construct contains the followingcomponents:

1. 5126::lexAop::DAM methylase;

2. [a promoter that is inducible by a hormone (auxin, salicylic acid),chemical safener and the like] ::lexA; and

3. a selectable marker, for instance which imparts herbicide orantibiotic resistance, or which effects complementation of amino acid ornucleic acid auxotrophs. When this construct is transformed into plants,the resulting phenotype is male-sterile in the absence of a chemicalinducer. But application of inducing agent at the appropriate timeresults in male-fertile plants, eliminating the need for geneticallycrossing plants that contain the sterility constructs with plants thatcontain repressor constructs in order to restore fertility. (See U.S.Ser. No. 07/848,465.) Examples of herbicide resistance genes include BARand PAT for glufosinate (bialophos) resistance.

When a construct of the present invention is linked with a selectablemarker such as a herbicide resistance gene, the resulting constructenables a method to destroy segregating male fertile plants by applyinga herbicide to the plants generated from crossing male-sterile plantswith pollen from male fertile plants. Only the male sterile plants willsurvive.

Another way to utilize the components of this system in a recombinantDNA construct used to transform a plant is to embed an operator capableof controlling expression of a DNA sequence (e.g., a lexA operator), ina tissue specific promoter (e.g., the anther-specific promoter 5126);the tissue-specific promoter operatively linked to a DNA sequence thatproduces a gene product which inhibits pollen formation or function,e.g., a dominant negative gene such as DAM-methylase. To embed such anoperator includes placing it (according to methods known to one skilledin the art) within, upstream or downstream of the nucleotide sequence ofthe promoters of the invention.

To reverse this effect, a plant transformed with such a construct ismated with a second plant that contains a second construct comprisingthe 5126 or other suitable promoter, including other anther-specificpromoters such as the 5126 deletion mutation promoters or constitutivepromoters, controlling the expression of a gene encoding a DNA-bindingprotein, e.g., the lexA gene which expresses the DNA binding proteinlexA, which is capable of binding to the operator of the firstconstruct. Specifically, the DNA-binding protein binds to the operatorof the first construct and represses expression, thus shutting offexpression of the DNA encoding a gene product which inhibits thefunction or formation of pollen and rendering a plant having both afirst and a second genetic construct, male-fertile.

In a specific embodiment, LexA repressor protein produced by the secondconstruct binds to the lexA operator embedded in the 5126 promoter inthe first construct and, as a consequence of binding to this region ofDNA, shuts off expression of the gene which inhibits pollen formation orfunction, e.g., a dominant negative gene such as DAM-methylase, andrenders the transformed plant male-fertile.

When a construct of the present invention is linked with a selectablemarker gene such as a herbicide resistance gene, the resulting constructenables a method to destroy segregating male fertile plants by applyinga herbicide to the plants generated from crossing male-sterile plantswith pollen from male fertile plants. Only the male sterile plants willsurvive.

According to another embodiment of the present invention, a geneticconstruct that has a methylase gene as the dominant negative geneoperably linked to a tissue-specific promoter, such as theanther-specific 5126 promoter, is suitable for the practice of thepresent invention. A method for altering the development of a plantrepresents an aspect of the present invention. Such a method preferablycomprises the steps of:

(a) transforming a plant with a genetic construct comprising a methylasegene and a suitable promoter; and

(b) growing the plant in an environment in which the methylase gene isexpressed, thereby altering expression of a gene, or genes, essentialfor a developmental process by methylating its promoter.

To produce a male-sterile plant, the promoter allows gene expressiononly in a specific tissue, preferably a tissue critical for pollenformation or function, such as in the tapetum, in the anther or in earlymicrospores. The construct may also include a methylase gene as the DNAsequence encoding a gene product capable of inhibiting pollen formationor function. A suitable methylase gene is a bacterial DAM (DNA adeninemethylating) gene. Bacterial sources include E. coli. The DAM class ofgenes methylates a N6 position of adenine in the nucleotide sequenceGATC. The construct includes a target DNA and is dominant negativebecause it represses the synthesis of mRNA by the target DNA.

A tissue-specific promoter is a promoter capable of controllingexpression of a DNA sequence, for example a gene, in a specific tissue.For causing reversible male sterility in plants, promoters that areactive in tissues directly or indirectly affecting pollen structureand/or function, are particularly suitable.

The search for tissue-specific promoters benefitted from the novelconcept in plant genetics, of subtracting mutant from normal plant mRNAto result in mRNA differing from the normal in areas of the genomespecifically related to the functions of interest in the presentinvention, anther development. An embodiment suitable for the presentinvention is an anther specific promoter, for example, the active DNAsequences of the novel plant promoter designated 5126.

Methods and compositions are described below for the production ofmale-sterile lines by the use of genetic constructs that include amethylase gene and a suitable promoter.

To correlate the insertion of a genetic construct of the presentinvention into a plant nuclear genome, with the male sterile phenotypeof the plant, Southern blots of DNA of plants were analyzed. By thisanalysis, male sterility was found to be correlated with the presence ofa genetic construct of the present invention.

In an embodiment of the invention, in order to destroy segregating malefertile plants so they do not grow in a field, a constitutive promoteris linked to a selectable marker and introduced into a plant with agenetic construct comprising a methylation gene regulated by a promoter.This system is useful when maintaining a sterile inbred line wherein amale fertile inbred plant is bred to a male-sterile plant of the sametype. Seed harvested from the female male-sterile plant will segregate1:1 for resistance to a selective agent. The plants may be sprayed withthe selective agent; consequently, only the plants that have maintainedthe selectable marker gene survive. These plants are those that weretransformed with the methylating construct.

The present invention also relates a male-sterile plant produced bymethods of the present invention, and to the seed of such plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 lists a nucleotide sequence comprising the region upstream fromthe coding region of the genomic clone for 5126, the nucleotide sequencecontaining sequences of the promoter elements of the 5126 promoter. Thecoding sequence for clone 5126 begins with the ATG start codon atposition 1488.

FIG. 2 presents a map of the DP5130 plasmid showing the NheI deletion ofthe maize 5126 promoter fused to the firefly luciferase gene.

FIG. 3 sets forth the relative activity of P5126 deletions. Coordinatesshown are relative to the translational start codon.

FIG. 4 provides information on tissue specificity of the 5126 promoterand deleted fragments of the promoter.

FIG. 5 is a graphical representation of stage specificity of the −503P5126 deletion used in the DP5814 plasmid: Pre=Premeiotic; Mei1=MeiosisI; Mei2=Meiosis II; Q=Quartet; QR=Quartet Release; EU=Early Uninucleate;EMU=Early-Mid Uninucleate; LMU=Late-Mid Uninucleate.

FIG. 6 presents a map of the DP5814 plasmid, which contains a 5126deletion promoter fused to E. coli DAM methylase and also contains thedouble CaMV 35S promoter, ADHI intron fused to the gene BAR and pinIIterminator.

FIG. 7 presents a map of the L87BspHI plasmid including the E. colilexA202 gene containing a mutagenized ATG codon within a novel BspHIrestriction site.

FIG. 8 presents a map of the L121 plasmid containing the double CaMV 35Spromoter, ADH1 intron fused to the lexA202 maize C1 gene hybrid andpinII terminator.

FIG. 9 presents a map of the DP5817 plasmid, containing the double CaMV35S promoter, ADH1 intron fused to the lexA202 gene and pinIIterminator.

FIG. 10 presents a map of the DP6232 plasmid which contains a minimalCaMV 35S promoter (−33) containing lexA binding site, ADH1 intron,firefly luciferase and pinII terminator.

FIG. 11 presents a map of the DP6509 plasmid which contains a lexAbinding site with minimal −33 CaMV 35S promoter, Adh1 intron,DAM-methylase and pinII terminator, and which also contains the 5126promoter fused to lexA202-C1 and a selectable marker construct, CaMV35S::BAR.

FIG. 12 is a bar graph illustrating lexA202-C1 activation and lexAmediated repression in maize embryogenic suspension cells, at varyingDNA doses (the numbers shown identify relative amounts of DNA).

FIG. 13 presents a map of the PHP6522 plasmid which contains the 5126deletion promoter fused to the E. coli lexA gene and also contains thedouble CaMV 35S promoter, maize ADH1 intron fused to the BAR gene andpinII terminator.

FIG. 14 presents a map of the PHP6555 plasmid which contains the maizeubiquitin promoter and intron fused to the E. coli lexA gene and alsocontains the double CaMV 35S promoter, maize ADH1 intron fused to theBAR gene and pinII terminator.

FIG. 15 presents a map of the PHP6520 plasmid which contains a lexAbinding site with a minimal −33 CaMV promoter, Adh1 intron,cornynebacteriphage diphtheria toxin A subunit and gene 7 terminator,and which also contains the 5126 promoter fused to lexA202-c1 and theselectable marker construct CaMV 35S::BAR.

FIG. 16 presents a map of the PHP8036 plasmid which contains the 5126deletion promoter, a lexA binding site with a minimal −33 CaMv promoter,Adh1 intron, E. coli Dam methylase and pinII terminator which alsocontains the selectable marker construct Ubiquitin:PAT.

FIG. 17 presents a map of the PHP8037 plasmid which contains the 5126deletion promoter, a lexA binding site with a minimal −33 CaMV promoter,E. coli Dam methylase and pinII terminator which also contains theselectable marker construct Ubiquitin:PAT.

FIG. 18 lists the DNA sequence of the 5126 cDNA. The putative start oftranslation of the cDNA sequence is at nucleotide position 73.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates the use of a genetic construct whichincludes a transcriptional activator and gene capable of acting on a DNAbinding site to activate a dominant negative gene, a dominant negativegene, and suitable promoters, including a tissue-specific promotercontrolling a gene acting on a DNA binding site, to affect plantdevelopment, for example, to cause male sterility. In transgenic plants,suitable dominant negative genes include cytotoxin genes, methylasegenes, growth-inhibiting genes. Dominant negative genes includediphtheria toxin A-chain gene (Czako and An, 1991), cell cycle divisionmutants such as CDC in maize (Colasanti et al., 1991) the WT gene(Farmer et al., 1994) and P68 (Chen et al., 1991). In an illustrativeembodiment, the DAM-methylase gene, the expression product of whichcatalyzes methylation of adenine residues in the DNA of the plant, isused. Methylated adenines will not affect cell viability and will befound only in the tissues in which the DAM-methylase gene is expressed,because such methylated residues are not found endogenously in plantDNA. A suitable system for DNA binding is the lexA-C1 system. Generally,the construct is exogenous and includes suitable promoters.

Altering development is particularly useful to produce a male-sterileplant. A method for producing a male-sterile plant is to transform aplant cell with a recombinant molecule (genetic construct) comprisingthe sense gene for the methylase protein. An appropriate promoter isselected depending on the strategy for developmental control. Forexample, a strategy is to express the methylase gene selectively inanther tissue by using an anther specific promoter. To produce amale-sterile plant, the transformed cell would be regenerated into aplant, pursuant to conventional methodology (see Materials and Methods).

In another embodiment of the present invention, a male-sterile plant isproduced by placing a methylase gene under control of a promoter that isexpressed selectively in cells critical to pollen formation and/orfunction.

“Exogenous” used herein denotes some item that is foreign to itssurroundings, and in particular applies here to a class of geneticconstructs that is not found in the normal genetic complement of thehost plant or is expressed at greater levels than in the endogenousstate.

A “suitable promoter” includes a tissue-specific or cell-specificpromoter that controls gene expression in cells that are critical forthe formation or function of pollen, including tapetal cells, pollenmother cells, and early microspores.

In an embodiment designed to affect cells selectively that are criticalto pollen development or function, a promoter that regulates geneexpression in a specific cell or tissue, such as a tapetal cell, is usedto control a gene encoding a DNA binding protein or a methylation sensegene.

A suitable promoter in this context is a tissue-specific regulatoryelement that effects expression only in tapetal tissue. Among suchsuitable promoters is the aforementioned 5126 promoter, derived from the5126 clone, which restricts expression of a DNA sequence to anthertissue. The 5126 promoter includes nucleotide sequences upstream fromthe coding region of the genomic clone for 5126, as shown in FIG. 1,which are capable of controlling or regulating expression of a DNAsequence in anther tissue. Deletion mutants of the 5126 promoter, suchas those characterized in Section (B) infra, are also suitable for usein the present invention in addition to specific regions of the 5126promoter nucleotide sequence which exhibit the desired selectiveexpression in anther tissue. Such specific regions of the 5126 promoterhave been characterized and are set forth in Section (B) infra. Othersuitable promoters include G9, SGB6, and TA39. Details of isolation anduse of TA39 promoters are presented in the materials and methods sectionherein.

For the present invention, the condition of “male sterility in a plant”means 100% sterility, with no viable pollen shed. The condition can beascertained by methodology well known to those skilled in the art,including such methods as determining pollen shed and germination tests.

An “anther-specific promoter” is a DNA sequence that directs a higherlevel of transcription of an associated gene in anther tissue than insome or all other tissues of a plant. Preferably, the promoter onlydirects expression in anthers. For example, the 5126 promoter isexpressed in anther cells. The anther-specific promoter of a genedirects the expression of a gene in anther tissue but not in othertissues, such as root and coleoptile. Promoters of this specificity aredescribed for example, in published European application 93810455.1, thecontents of which are hereby incorporated by reference.

An “operator” (or “DNA binding site”) is a DNA molecule that is locatedtoward the 5′ end of a structural gene and that contains a nucleotidesequence which is recognized and bound by a DNA binding protein that haseither activation or repression function. The binding of a repressorprotein with its cognate operator results in the inhibition of thetranscription of the structural gene. For example, the lexA gene encodesa repressor protein that binds to the lexA operator.

An “isolated DNKA molecule” is a fragment of DNA that is not integratedin the genomic DNA of an organism. Isolated DNA molecules may bechemically-synthesized.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A “cloning vector” is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

An “expression vector” is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

The following examples are set forth as representative of specific andpreferred embodiments of the present invention. These examples are notto be construed as limiting the scope of the invention in any manner. Itshould be understood that many variations and modifications can be madewhile remaining within the spirit and scope of the invention.

EXAMPLE 1 Isolation and Characterization of the 5126 Promoter

(A) METHODOLOGY

Methods used for isolation of an anther specific promoter were novel formaize. The subtraction method of gene isolation only was useful afterdetermination of the time in development that a suitable anther specificgene would be expressed, so that mRNA could be collected before andafter that development threshold, to isolate a suitable gene.

Extensive comparisons of development of anthers from male-fertile maizewith anthers from male-sterile maize suggested that anther mRNAsubtraction at a time just before microspore degeneration would yieldunique, anther-specific mRNAs. Total RNA was isolated from anthers frommale-sterile plants just before microspore breakdown. With the dominantmale-sterile mutant Ms44, this meant collecting anthers that were on orabout the quartet stage of microsporogenesis. Anthers from fertilesibling plants also were collected at this stage. Male fertile and malesterile plants were collected as a source of mRNA.

(1) RNA Isolation: was performed by the guanidine isothiocyanate methodknown to those of skill in the art.

(2) mRNA Isolation: was accomplished by means of an oligo dT column byInvitrogen.

(3) cDNA Library construction: Libraries were made from tassel mRNA frommaize stocks of a dominant male sterile mutation (Ms44) and its malefertile sibs (ms44) (available from Maize Stock Center, University ofIllinois). The libraries were made by Invitrogen who used thebi-directional cloning method with the pCDNAII vector and cloning atBstXI sites.

(4) Subtraction: Subtraction was done as described in the “TheSubtractor I” instruction manual from Invitrogen version 2.3. usinglabelled cDNA from the male sterile dominant library as the driver, andunlabelled male fertile library as the tester (See Materials andMethods). This new library was labelled #5 and was expected to containunique male fertile cDNA's.

(5) Unique Clones: Clones were isolated randomly from library #5 andinserts were gel purified and random hexamer labelled with P32 as wellas slot blotted onto nitrocellulose. Duplicate clones were avoided bycross-hybridization. 5126 was one clone selected from the subtractedlibrary #5. It was hybridized with non-tassel cDNA to ensure antherspecificity of the clone.

(6) Full-Length cDNA Isolation: To obtain a full length 5126 cDNA apartial 5126 cDNA clone was isolated and sequenced using the m13universal primer 5′TGTAAAACGACGGCCAGT 3′ (M13 UP) and the ml3 reverseprimer 5′CAGGAAACAGCTATGACC 3′ (M13 RP). This partial 5126 cDNA clonecontains an insert of 594 bases which includes a polyA+ tail of 27nucleotides. Total RNA and mRNA were isolated for library construction.The cDNA library was made by Stratagene using the Uni-Zap XR directionalcloning system (EcoRI to XhoI). 1×10⁶ PFU were screened with an EagIfragment from the partial 5126 cDNA to obtain a full length 5126 cDNA.ER1647 (NEB) was used as the host bacterium. Ten positive clones werepurified to homogeneity. Plasmids were made by in vivo excision of thepBluescript SK(−) phagemid from the Uni-Zap XR vector (Stratagene LambdaZap Instruction Manual, page 14). Sequencing was done by United StatesBiochemical Company on clone p5126-5; the sequence is set forth in FIG.18. Both strands were entirely sequenced and agreed with the sequence ofthe partial cDNA. A Northern blot was done with the partial cDNA whichindicated a transcript length of about 1.5 Kb. p5126-5 has a length of1.485 Kb, which indicates it represents a full or nearly full lengthcDNA.

(7) Genomic Isolation: A genomic library was constructed from maizeinbred line B73 DNA was partially digested with Sau3A1 and cloned intothe BamHI site of λ DASH II (Stratagene). 1×10⁶ PFU were screened withan EagI fragment from the partial 5126 cDNA. ER1647 (NEB) was used asthe host bacterium. Three clones were isolated to homogeneity afterthree rounds of screening. DNA from these λ clones was isolated using amethod reported by Bellomy and Record, (1989) and restriction sites weremapped. All three clones were identical, spanning approximately 18 Kb.

(B) CHARACTERIZATION OF PROMOTER 5126

(1) Northern analysis:

An EagI fragment derived from the partial 5126 cDNA was used to probe aNorthern membrane containing maize polyA+ mRNA from etiolated leaves,roots, and green leaves from 6 day old seedlings, tassels withpremeiotic stage anthers, tassels with meiotic stage anthers, tasselswith quartet through uninucleate microspore stage anthers and earshoots. The EagI fragment was labeled with horseradish peroxidase usingthe Enhanced Chemiluminescence (ECL) system from Amersham. Hybridizationof the probe and membrane washes followed the manufacturer's protocolthe ECL system. The cDNA probe hybridized to transcripts approximately1.6 kb, present only in mRNA from tassels with quartet throughuninucleate microspore stage anthers.

(2) Sequence analysis:

Three genomic clones in lambda DASHII which hybridized to the 5126 cDNAprobe were isolated. These clones are 5125.4, 5126.5 and 5126.8.

From one of the genomic clones, 5126.8, a HindIII fragment ofapproximately 5 kb was isolated and subcloned into the HindIII site ofthe vector, BluscriptII KS+ (Stratagene). Two plasmids, DP4769 andDP4770, were generated containing the HindIII fragment inserted in twodifferent orientations. The plasmids DP4769 and DP4770 were partiallysequenced for one strand using the m13 universal primer, m13 reverseprimer and with the oligonucleotide 5′CCTTCATCAGCTTCTGGCAG 3′ (DO776).The sequence of DO776 was derived from the sequence of the 5′ portion ofthe 5126 cDNA insert. A double strand sequence of DP4770 was obtained by“primer walking” with the following oligonucleotides,5′AGATCTCGGCCAGGCCCTTG 3′ (DO990), 5′GAGTTGATGAAGTGA 3′ (CWG4770),5′GAGATCAATCAGCTAGAGG 3′ (PG2-2), and 5′TAAACCTAAGGCC 3′ (PG2-3). Thesequence of DP4770 from the HindIII site to the region immediatelyadjacent to the DO990 sequence is 1594 bases.

A SacI fragment of approximately 6 kb long was isolated from the genomicclone 5126.8 and inserted into the SacI site of the vector BluscriptIIKS+ (Stratagene). Two plasmids, DP5053 and DP5054, were generated withthe SacI fragment inserted in two different orientations. The SacIfragment overlaps by 1207 base pairs with the HindIII fragment used forDP4769 and DP4770. This overlap is 5′ of the region of DP4769 and DP4770with homology to the cDNA insert of 5126. The sequence of 2106 bases forDP5053 was obtained by primer walking with the same oligonucleotidesused for sequencing DP4770 and also with oligonucleotide5′AATAGCCTAATTTATTAG 3′ (PG2-4), oligonucleotide 5′ACATGTTTCAAGTTCAA 3′(PG2-5), oligonucleotide 5′CTTGTCAGAAGTTGTC 3′ (PG2-5C) andoligonucleotide 5′CAACCATTACCGATGAA 3′ (PG2-6C).

5′RACE was used to obtain additional coding sequences for the 5126 gene.5′RACE primer extension was performed using the 5′RACE system (GibcoBRL) with the oligonucleotide 5′ACGAGCGGACGCACGACAG 3′ (DO1168), derivedfrom the sequence of DP4770, for primer extension with polyA RNA frommaize tassels. The nested primer 5′TCCGTCGCCATCTGCGTCAC 3′, also fromthe DP4770 sequence, and the anchor primer5′CACGCGTCGACTAGTACGGGIIGGGIIGGGIIG 3′ (DO805) (modified from the anchorprimer included in the 5′RACE system) were used for PCR amplificationwith TaqI DNA polymerase (Perkin Elmer). The 5′ RACE product wassubcloned into the pT7Blue(R) vector (obtained from Novagen). A clonecontaining the PCR product was named CGR3B. This plasmid was sequencedusing DO805, DO1398 and m13 universal primers. The 5′RACE PCR insert is412 bases long. There are polymorphisms between the near full lengthcDNA of the new A632 library, compared to the genomic clone from the B73library and the original clone.

The sequence from CGR3B matches 586 bases of DP4770 with a 123 baseintron present in the genomic sequence. The intron contains the highlyconserved intron splice site motifs (5′ GT and 3′ AG). A putative startcodon is seen which is in frame with the rest of sequence. This startcodon has a reasonable start codon motif (CGATGG). Immediately upstreamof this putative start codon, the sequence of CGR3B is relatively ATrich which is characteristic of 5′-untranslated cDNA sequences. Thereare 90 nucleotides in CGR3B upstream of the putative start codon whichis a reasonable length for 5′ untranslated regions in plants. Inaddition, the 5′ most end of the CGR3B sequence homology in DP4770 is 35bases downstream of a reasonable TATA box (TATATA). The 5126-5 sequenceoverlaps the sequence of CG3RB, with CGR3B having an additional 43 basesupstream.

This size correlates reasonably well with the transcript size estimatedfrom northern hybridization of approximately 1.6 kb.

(3) Site-directed mutagenesis

Site directed mutagenesis (Su and El-Gewely, 1988) was used to create anNcoI site in DP5053 at the putative translational start codon with theoligonucleotide 5′GCTGCTCACCATGGCAAAGCAAC 3′ (DO1398) to create DP5055.

(4) Reporter constructs

A ScaI-NcoI fragment of approximately 4 kb, 5′ of the 5126 codingregion, was isolated from DP5055 and combined with a SmaI-NcoI fragmentof DP1672 which contains the vector, the firefly luciferase region andthe untranslated region of the proteinase II gene (pinII), to make thereporter construct DP5062. Deletions into the 5′ end of the 5126promoter fragment of DP5062 were prepared by removing sequences from theHindIII site in the polycloning region to the HindlII site 587 basesupstream of the ATG start condon (DP5121), or removing the sequence fromthe PstI site in the polycloning region to the PstI site 170 basesupstream of the ATG start codon (DP5122). Additional deletions from the5′-end of the promoter fragment were generated by making use of the SphIsite 855 bp upstream of the translational start codon, the NdeI site 503bp upstream of the start codon, or the KpnI site 216 bp upstream of thestart codon. DO5062 was digested with SphI or NdeI, blunted with T4 DNApolymerase, and digested with NcoI after inactivating the polymerase.The resulting promoter fragments were cloned to the SmaI/NcoI fragmentof DP1672, containing the vector of the luciferase reporter fused to thePinII 3′ region. This gave rise to DP5131 (SphI deletion) and DP5130(NdeI deletion) (FIG. 2). The KpnI deletion (DP5164) was obtained by athree-piece ligation of (1) the KpnI/ClaI fragment containing thepromoter/luciferase junction, (2) the ClaI/AlwNIluciferase/PinII-3′/vector fragment, and (3) the AlwNI/KpnI fragment ofthe remaining vector piece from DP5062.

(5) Transient assays

FIG. 3 shows the specific activity of luciferase obtained in anthers atthe quartet to early uninucleate stage, when transformed with the fulllength 5126 promoter-luciferase construct (DP5062) or promoter deletionderivatives. Essentially full activity is observed in deletions up tothe NdeI site 503 bp upstream of the translational start codon, butnearly all activity is lost upon deletion to the KpnI site 216 bpupstream of the start codon. No activity remains upon deletion to thePstI site 170 bp upstream of the start codon. Thus, a critical elementis likely to occur between 170 and 503 bp upstream of the translationalstart codon.

FIG. 4 shows the luciferase specific activity obtained in anthers,coleoptiles, roots and embryogenic suspension culture cells for theoriginal 5126 promoter fragment reporter construct (DP5062) and the twokey deletions (DP5130 and DP5164) compared to positive andtissue-specific controls (DP1528, containing a luciferase reporter genedriven by a “constitutive” CaMV 35S promoter, and DP2516, containing aluciferase reporter driven by an anther-specific promoter SGB6).Tissue-specificity, observed for the full-length promoter fragment, wasmaintained in the NdeI deletion.

FIG. 5 shows the timing of anther activity of the 5126 (−503) promoter.This deletion promoter is most active in early uninucleate microsporestages, although activity spans meiotic stages through themid-uninucleate microspore stage.

EXAMPLE 2 Construction of DAM-methylase Plasmids

A DAM-methylase gene was obtained from E. coli. A methylase gene derivedfrom any plant is also suitable.

The DAM-methylase gene (nucleotides 195-1132 from Brooks, et al., 1983)was modified by site-directed mutagenesis (Su and ElGewley, 1988) and aSmal site was introduced at nucleotide 186, nine nucleotides 5′ to theinitiating codon ATG. DP5814 (FIG. 6) is a plasmid used in maizetransformation which contains the anther-specific DAM-methylase gene incis with a constitutively expressed BAR gene. This plasmid wasconstructed by ligating the 500 bp XhoI/NcoI fragment containing theNdeI-NcoI deletion of the 5126 anther-specific promoter region fromDP5130 (FIG. 2) to a 1.0 kb SmaI/BamHI fragment containing the modifiedDAM-methylase sequences described above. The NcoI site contained on theXhoI/NcoI 5126 promoter fragment was filled in with dNTPs using T4 DNApolymerase (Boehringer-Mannheim) according to established protocols(Sambrook et al., 1989) to generate a blunt-end for cloning. Thepromoter/gene junction resulted in the addition of 3 N-terminal residuesencoded by the following sequence (the initiating MET of the nativeDAM-methylase gene is underlined and corresponds to nucleotides 195-197in Brooks et al., 1983):

5′CCATGGGGACAATG 3′ The DAM-methylase expression is terminated byligating the 320 bp BamHI-NotI fragment that contains the 3′ PinIIsequences from the potato proteinase inhibitor II gene (nucleotides2-310, from An et al., 1989). This chimeric gene contained on a 1.6 kbXhoI-NotI DNA fragment was cloned into the XhoI-NotI restriction site ina monocot expression plasmid that contains the enhanced cauliflowermosaic virus 35S promoter (nucleotides −421 to +2, repeating −421 to −90in tandem, Gardner et al., 1981), the tobacco mosaic virus (TMV) leader(79 bp HindIII-SalI fragment, as reported by Gallie et al., 1987), a579-bp fragment containing the intron 1 from the Adh-S allele of themaize alcohol dehydrogenase gene (Dennis et al., 1984), the BAR genewhich encodes for the enzyme phosphinothricin acetyl-transferase(nucleotides 160-704 from Thompson et al., 1987, where the nucleotide160 was changed from a G to an A to generate a MET initiation codon) andthe termination sequences from the potato proteinase inhibitor II gene(nucleotides 2-310, from An et al., 1989), in a pBluescript (Stratagene)backbone.

EXAMPLE 3 Production of a Male-Sterile Plant

Plants were transformed with DP5814. DP5814 contains the Nde1 deletionderivative of the 5126 promoter fused to the E. coli DAM-methylase geneand the PINII terminator. This plasmid also contains the double 355cauliflower mosaic virus promoter fused to the BAR gene. (Thompson etal., 1987).

Construct PHP6522 (FIG. 13) is identical to that described for DP5814with the exception that the coding sequences of the Dam methylase genewas replaced by the lexA coding region from amino acid 1 to 202(Golemis, 1992).

Construct PHP6555 (FIG. 14) is identical to that described for PHP6522with the exception that the 5126 promoter was replaced by the maizeubiquitin promoter and intron which is contained on a 1.9 kB PstI DNAfragment.

DP5814 was bombarded into Hi Type II (B73×A188) (Armstrong, 1991) calluscell-lines from which Bialophos-resistant plants were regenerated. Toserve as controls for male-fertility, untransformed plants were alsogenerated. Transgenic and control calli were analyzed by PCR.

A transgenic plant containing a methylase gene construct can beregenerated from a culture transformed with that same construct, so longas the plant species involved and the type of culture used aresusceptible to regeneration. “Culture” in this context comprehends anaggregate of cells, a callus, or derivatives thereof that are suitablefor culture.

A plant is regenerated from a transformed cell or culture, or from anexplant, by methods disclosed herein that are known to those of skill inthe art. Methods vary according to the plant species. Seed is obtainedfrom the regenerated plant or from a cross between the regenerated plantand a suitable plant of the same species using breeding methods known tothose of skill in the art.

EXAMPLE 4 Effect of 5126::DAM-Methylase on Fertility of Maize Plants

Regenerated maize plants transformed with the DP5814 construct wereanalyzed by PCR for the presence or absence of the DAM-methylase codingregion and scored for their ability to generate fertile pollen.

The polymerase chain reaction (PCR), which is well-known to those ofskill in the art, was used to determine the presence of the E. coliDAM-methylase gene. The oligonucleotides used were DO1266 and DO1267:

The oligonucleotides have the following sequences:

DO1266

5′-ATG AAG AAA AAT CGC GCT TTT TTG AAG TGG GC-3′

DO1267

5′-TCA CCC AGG CGG GCA AAA TCA GCC GAC A-3′ These oligos were employedas primers in PCR to amplify the E. coli DAM-methylase genespecifically.

Twenty-five independent primary transgenic maize plants that were PCRpositive for the DAM-methylase gene were analyzed. Twenty-two of theseDAM-methylase PCR positive plants were male-sterile. Southern analysisconducted on these plants detected the presence of single-copy tomultiple copy insertion events. Microscopic examination of pollendevelopment in these male-sterile plants as compared to either PCRnegative or untransformed plants revealed that premeiotic and meioticmicrospores can be observed in all plants, however quartet microsporeshave not been observed in any of the anthers derived from plants thatare PCR positive for the DAM-methylase gene and are male-sterile. Thisbreakdown of microspore development is consistent with the observationthat luciferase activity can first be detected at a similar stage ofdevelopment when expressed under the control of the 5126NdeI deletionpromoter, suggesting that expression of the DAM-methylase gene duringearly microspore development interferes with normal pollen formation.

Male-sterile maize plants were pollinated with pollen derived fromuntransformed maize plants, the seed was germinated and resulting plantswere analyzed for co-segregation of herbicide resistant male-sterileplants with the presence of the 35S: Bar - 5126:DAM-methylase constructto establish a correlation between the presence of the methylase geneand male-sterility. Southern analysis of T1 populations derived from 13independent male-sterile T0 events has revealed that all of themale-sterile bialophos resistant plants contained the E. coliDAM-methylase and BAR genes whereas male fertile, bialophos sensitivesegregants did not contain these genes.

Similar to the observations made in the T0 plants, microsporedevelopment breakdown occurred between meiosis I and quartet stages.

EXAMPLE 5 Southern Blotting to Correlate the Male Sterile Phenotype in aPlant with the Insertion of a Genetic Construct Capable of Methylation

Nine mls of CTAB extraction buffer (100 mM Tris pH 7.5), 1% Hexadecyltrimethyl-Ammonium bromide, 0.7M Sodium chloride, 10 mM EDTA) were addedto 300mg of lyophilized leaf tissue, vortexed and incubated at 65° C.for 1 hour. Five mls of a chloroform/octanol (24:1) solution were addedand mixed for 5 minutes. Extracts were spun for 30 minutes at 2500 rpm.The top layer was removed and placed in a new tube, and 11 mls of CTABprecipitation buffer (same as CTAB extraction buffer minus the sodiumchloride) were added, inverted and allowed to stand for 30 minutes. Thesample was spun for 10 minutes at 2000 rpm. To resuspend the pellet, 2mls of 100 mM Tris (pH 7.5), 10 mM EDTA, 0.7M NaCl were added and heatedfor 15 minutes at 60° C. 10 μl of RNAseA (10 mg/ml) were added andincubated for 30 minutes at 37° C. Five ml of cold 100% ETOH is added tothe tube and mixed gently, the DNA is hooked out using a bent 9 inchPasteur pipet, placed into a tube that contains 76% ETOH, 0.2M sodiumacetate and allowed to sit for 20 minutes. The DNA is transferred to anew tube that contains 76% ETOH, 0.2M ammonium acetate for 1 minute,wiped dry and resuspended in 300μl of TE (10 mM Tris [pH 7.5], mM EDTA).5 μg of genomic DNA digested with restriction endonucleases waselectrophoresed on 0.8% agarose gels containing Tris-acetate buffer; gelwas prepared for transfer to the membrane by incubating for 20 minutesin 500 mls of 0.25M HCl, 40 minutes in 500 mls of 0.4M NaOH, 0.6M NaCland 30 minutes in 0.5M Tris (pH 7.5), 1.5M NaCl. Transfer was done byusing 25 mM sodium phosphate buffer, pH 6.5 onto Amersham Nylon FPmembrane. After transfer, membrane was baked at 80° C. under vacuum.Prior to the first use of the membrane, it is incubated at 65° C. in asolution containing 0.1×SCP (1×SCP; 0.1M NaCl, 16 mM sodium phosphate,pH 7.0) and 0.1% SDS for 30 minutes. P32-dCTP labelled DNA probes weregenerated with a random primer-labelling kit supplied by Amershamaccording to the manufacturers instructions. To generate theDAM-methylase specific probe, the 635 bp BamHI DNA fragment was isolatedfrom DP5814 and labelled. To generate a BAR-specific probe, a 560 bpNcoI-BamHI DNA fragment was isolated from DP5814 and labelled. Thelabelled probe was denatured for 10 minutes at 95° C., added to thefilter in 20 mls of hybridization buffer (0.1×SCP containing 0.1×Dextransulfate) and incubated at 65° C. overnight. The filter was washed 3times with 0.1×SCP containing 0.1% SDS at 65° C. The filter was exposedto X-ray film with a screen (Dupont) at −70° C.

EXAMPLE 6 Construction of Transient Assay Plasmids

A HindIII/Xhol fragment containing the LexA202 gene (nucleotides734-1406 in pEG202 in Golemis and Brent, 1992) was cloned intopBluescriptSK+(Stratagene) to generate plasmid L87. Site-directedmutagenesis (Su and El Gewley, 1988) of this plasmid using the oligoDO2326:

5′CCGTTAACGCTTTCATGACGCCCGGAATTAAGC 3′ resulted in the introduction aBspHI site at the initiating ATG of the LexA-202 reading frame(nucleotide 754, Golemis and Brent, 1992) generating the plasmidL87BspHI (FIG. 7). A chimeric gene containing the LexA sequencesencoding residues 1-202 on a BspHI/EcoRI fragment from L87BspHI wasfused in-frame with an EcoRI/Hpal fragment residues 144-273 from themaize C1 described above into a monocot expression plasmid containingthe enhanced cauliflower mosaic virus 35S promoter (nucleotides −421 to+2, repeating −421 to −90 in tandem, Gardner et al., 1981), the tobaccomosaic virus (TMV) leader (79 bp HindIII-Sa1I fragment, as reported byGallie, et al., 1987), a 579-bp fragment containing the intron 1 fromthe Adh-S allele of the maize alcohol dehydrogenase gene (Dennis et al.,1984), and the termination sequences from the potato proteinaseinhibitor II gene (nucleotides 2-310, from An et al., 1989), in apBluescript backbone generating plasmid L121 (FIG. 8).

The construct DP5817 (FIG. 9) contains the enhanced CaMV promoter, TMVleader Adh intron and the PinII termination sequences described above.The sequences coding for residues 1-202 of the LexA protein carried on aBspHI/SmaI fragment from L87BspHI (nucleotides 754-1382 in pEG202 inGolemis and Brent, 1992) were cloned downstream of the Adh intronreplacing the LexA-C1chimeric gene found in L121.

The reporter plasmid, DP6232 (FIG. 10) contains three tandemly repeatedlexA DNA binding sites carried on the complementary oligonucleotides,DO2448 and DO2449, with the following nucleotide acid sequences.

DO2448:

5′GATCTACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACTGCTGTATATAAAACCAGTGGTTATATGTACAGTACGGATG 3′ D 0 2 4 4 9 :

3′ACGACATATATTTTGGTCACCAATATACATGTCATGACGACATATATTTTGGTCACCAATATACATGTCATGCCGATG 5′ The oligos were annealed and cloned as aBgIII/NdeI fragment upstream of a truncated CaMV promoter (nucleotides−33 to +2; see Gardner et al., 1981), the TMV leader, ADH intron, thecoding region of the firefly luciferase gene (+53 to +1708, dewet etal., 1987), and the PinII termination sequences in a pBluescriptback-bone.

Construct DP6509 (FIG. 11) is a plasmid containing three chimeric genesdesigned for expression in maize plants. The plasmid also contains thelexA binding sites upstream of a truncated CaMV promoter, the TMV leaderand ADH intron and PinII terminator as described for DP6232 with theDAM-methylase gene, maintaining the 9 bp addition as described above inplace of the luciferase coding sequences. The gene sequences encodingthe anther-specific transcriptional activator 5126::LexA-C1 are locatedimmediately downstream of the DAM-methylase reporter gene describedabove. This gene contains the XhoI/NcoI fragment carrying the 5126promoter sequences from DP5130, the LexA202-C1 chimera and PinIIsequences described for L121. The third gene encoded by this plasmidcontains the enhanced CaMV promoter, TMV leader, Adh intron, BAR codingsequences and the PinII terminator on a pBluescript backbone asdescribed for DP5814.

Construct PHP6520 (FIG. 15) is the same as that described for PHP6509with the exception that the coding sequences of the Dam Methylase geneand pinII terminator were replaced by the diphtheria toxin coding regionand gene 7 terminator (Czako and An, 1990).

Construct PHP8036 (FIG. 16) contains a the 5126 promoter from positions−503 to −134, fused to the lexA binding site upstream of the minimal −33CaMv promoter, the TMV leader, ADH1 intron the coding region of Dammethylase and the pinII terminator as described for DP6509. The plasmidalso contains the selectable marker construct Ubi-Pat, which wasconstructed by fusing a 1.9 kB maize ubiquitin promoter and intron tothe modified phosphinothricin-N-acetyl-transferase gene (Pat) fromStreptomyces viridochromagenes and the nopaline-synthetase gene (Droge,et al.).

Construct PHP8037 (FIG. 17) is identical to PHP8036 with the exceptionthat the maize AdhI intron contained within the 650 bp SalI/BamHI DNAfragment was removed from the 5126:lexA:Dam methylase portion of theplasmid.

EXAMPLE 7 Expression of a Luciferase Reporter Containing lexA BindingSite Upon Transient Co-Expression of Either lexA-C1, lexA or Both

Experiments were conducted to address two questions. First, can thebacterial DNA binding protein lexA promote and enhance gene expressionin plant cells? Second, does co-expression of the lexA protein with thetranscriptional activator lexA-C1 result in the repression ofactivator-mediated gene expression.

The lexA protein would bind to a region of DNA containing the lexA DNAbinding site (“lexA operator”) but would not recruit the necessary plantderived transcriptional components to initiate mRNA synthesis. But ithas been shown that juxtaposition of protein regions that can act astranscriptional activators to DNA binding proteins will result inincreased expression of the reporter gene (Ruden et al., 1991). To testthe ability of the lexA gene to promote expression of a reporter gene inmaize cells, a region of the maize C1 gene (Goff et al., 1991) encodinga transcriptional activation domain was fused in-frame with the regionof DNA that corresponds to the DNA binding protein lexA, to generate thehybrid gene, LexA202-C1. The hybrid gene was placed under thetranscriptional control of the constitutive promoter 35S to generateplasmid L121 as shown in FIG. 8.

This construct was co-bombarded at varying amounts into maizeembryogenic suspension cells with a constant amount of a luciferasereporter gene that contains the lexA binding site, plasmid DP6232. Asshown in FIG. 12, the reporter alone yields very low luciferase activity(fourteen light units per microgram total protein (14 lu/μg), howeverhigh luciferase activity (>9000 lu/μg) is detected when the lexA-C1transactivator is co-bombarded at amounts greater than 5 ng per shot.

To determine if the lexA protein will repress the high level ofluciferase expression, the plasmid DP5817 which contains a 35S:lexAconstruct as shown in FIG. 9 was co-bombarded with DP6232 and L121,varying the amounts of L121 or DP5817. As shown in FIG. 12, addition ofDP5817 to treatments containing the lexA-C1 construct and reporterresults in reduced luciferase activity. Together these data suggest thatin maize embryogenic suspension cells enhanced expression of a genecontaining a lexA DNA binding site is detected when the lexA-C1 fusionprotein is co-expressed and that this expression may be repressed by thelexA protein.

EXAMPLE 8 Reversion to a Male-Fertile Plant

In accordance with the present invention, there are several strategiesto produce reversion of a male-sterile to a male-fertile plant. Acascade effect wherein a promoter, such as the tapetal specific promoter5126 is fused to the transcriptional activator LexA-C1 gene (hereincalled 5126::LEXA-C1) where the LexA portion of the gene encodes thebacterial LexA protein that binds to a region of DNA called the LexAoperator (LexAop) and the C1 portion of the gene encodes the maize C1protein that interacts with the maize transcriptional machinery topromote transcriptional activation of genes that contain the LexAopwithin the context of a minimal promoter element, for example theminimal 35S promoter.

To generate a male-sterile maize plant the DAM-methylase gene is placedunder the control of the LexAop fused to the minimal CAMV 35S promoter.Contained on the same plasmid is the 5126::LexA-C1 region and aselectable marker, 35S:BAR (FIG. 11, DP6509). Introduction of thisconstruct renders the plants male-sterile due to the expression of theDAM-methylase gene in the anther. LexA-C1 is regulated by the 5126promoter.

In order to restore fertility to the male-sterile 5126:LexA-C1,LexAop::DAM-methylase containing plants, such plants are crossed toplants that contain the 5126 promoter or other suitable promoters fusedonly to the LexA DNA portion. The presence of a genetic construct whichincludes 5126:LexA is consistent with male fertility. In the presence ofa gene that expresses a protein that binds to the LexAop but does notactivate transcription of the DAM-methylase gene, synthesis of aDAM-methylase protein is repressed thus the plant is male-fertile.

Transgenic maize plants were generated as described herein to containplasmids PHP6522, PHP6555 and PHP6520. Of the transgenic events thatgenerated transgenic maize plants containing the male-sterilityconstruct PHP6520, 5 events were determined to be male sterile plants inthe T0 generation and 3 events were determined to be male fertile. 3 ofthe male sterile events were analyzed in the T1 generation forcosegregation of the male-sterile phenotype with Ignite resistance. Theresults are shown in Table 1:

TABLE 1 Ignite-resistant Ignite-sensitive Event Male Sterile Plants MaleFertile Plants 937.59.35.2 17 13 937.63.25.1 2 28 937.59.35.1 1 0

The male-sterile events 937.59.35.2 and 937.63.25.1 were crossed byusing pollen derived from plants that contain the lexA gene under thecontrol of either the Ubiquitin promoter (PHP6555) or the antherspecific promoter (PHP6522), respectively. The result is that plantscontaining both the sterility construct (PHP6520) and the repressorconstruct (PHP6522 or 6555) will be male-fertile, whereas plants thatcontain only the sterility construct PHP6520 will be male-sterile.

Transgenic events were generated as described supra using constructscontaining a modified version of the 5126 promoter (the nucleotidesequence from positions −503 to −134 relative to the start codon atposition 1488, as shown in FIG. 1) which has embedded the lexA bindingsite juxtaposed to the minimal CaMV promoter (PHP8036 and PHP8037).Introduction of those constructs renders the resultant plantsmale-sterile due to expression of the DAM-methylase gene. Suchmale-sterile plants containing either PHP8036 or PHP8037 are crossed toplants that express the lexA repressor in a constitutive (PHP6555) ortissue specific (PHP6522) fashion. The result is that plants containingboth the sterility construct (PHP8036 or PHP8037) and the repressorconstruct (PHP6522 or PHP6555) will be male-fertile, whereas plants thatcontain only the sterility constructs PHP8036 or PHP8037 will bemale-sterile.

MATERIALS AND METHODS

Subtraction Probe Procedure (from Invitrogen):

Generation of a subtraction cDNA probe was accomplished in a similarmanner to the method for generation of a subtraction library. Adiagrammatic outline of the method is shown below. In this scheme,labelled cDNA is first synthesized from the induced (message +) pool ofmRNA. The resulting cDNA-RNA hybrid is alkali treated to remove thetemplate mRNA and then hybridized to an excess of photobiotinylated mRNAfrom pool B (message −). The resulting photobiotinylated RNA/cDNAhybrids are complexed with free streptavidin and removed from thehybridization mixture by selective phenol/chloroform extraction. As inthe subtraction library procedure, the streptavidin-photobiotinylatednucleic acid complex is extracted leaving the unhybridized (induced)cDNAs behind. The resulting subtracted cDNA probe can be used directlyin hybridization blots or for screening libraries.

The use of a subtraction cDNA probe improves the chances of identifyingcDNA clones that correspond to tissue specific, rare transcripts. In atypical cDNA probe, the representation is proportional to mRNAabundance. By enriching the cDNA probe for sequences specific to adifferentially expressed gene, the probe becomes more specific for theintended clone which simplifies the screening of libraries. Asubtraction cDNA library can be used in conjunction with a subtractedprobe to identify cDNA clones representing low abundance mRNAs unique toa particular tissue or induced cell state. The advantage of using asubtracted cDNA library instead of a non-subtracted cDNA library is thatfewer clones have to be screened.

Methods for transient assay:

Maize embryogenic suspension cell cultures were derived from immatureembryos, maintained in liquid suspension as described (Bowen, 1992) andsubcultured every 3 to 4 days. Cells were harvested 2 days aftersubculture and, prior to bombardment, treated overnight in growth mediumcontaining 0.25M mannitol at a density of 50 mg/ml. For eachbombardment, 25 mg of cells was placed on filter paper premoistened with1 ml of growth medium. 3 μg of reporter plasmid DNA (DP6232) and varyingamounts of DP5817 and/or L121 (0.01-3 μg) was precipitated on 0.75 mg of1.8-μm tungsten particles and the cells were bombarded with one-sixth ofthis mixture using a PDS1000 helium gun, according to the manufacturer'sinstructions (DuPont). After 24 hours, the cells were harvested andtransferred to 1.5 ml screw cap microcentrifuge tubes and maintained at4° C. throughout all of the remaining procedures. Samples werehomogenized in 0. ml GUS lysis buffer (Rao and Flynn, 1990: modified bythe omission of all detergents) and cleared by centrifugation.Luciferase assays were performed as described by Callis et al., (1987)using a 10-sec integration time on a luminometer (Model 2010; AnalyticalLumenescene, San Diego, Calif.). Protein concentration was determinedusing a BioRad protein assay kit. Extracts were generally 0.75-1.5 μg ofprotein per of extract. Luciferase specific activity (1 μ/μg) wascalculated by measuring the luciferase light units in 25 μl of extractand the value corrected for the corresponding protein concentration perμl of extract. Luciferase activities shown in Table 1 are expressed asan average of three bombardments of each treatment.

Isolation of TA39 Genomic Clones Comprising Sequences Homologous toMicrospore-Specific mRNA; TA39 Promoters

This example provides methods of isolation of genomic DNA clonescomprising sequences homologous to any microspore-specific mRNA forwhich a nucleic acid probe is available. The approach described isuseful for isolating microspore-specific regulatory sequences from anyplant species which has microspore-specific mRNA that is homologous tosuch an available probe.

A tobacco anther-specific cDNA clone, TA39, was obtained from Dr. RobertGoldberg of UCLA. TA39 hybridizes to mRNA from anthers in a similartemporal pattern as seen with several tapetum-specific transcripts(Kultunow et al., 1990). In situ hybridizations showed that TA39 ispresent at low levels in microspores and connective tissue during stage−1 to +1 and then at higher levels in the tapetum from stage 1 through 6(Goldberg et al., 1993).

A genomic library of a selected plant, for instance a commerciallyavailable library of DNA fragment from N. tabacum, var. NK326 (ClontechLaboratories, Inc., Palo Alto, Calif.; catalog FL1070D), partiallydigested with MboI and cloned into the plasmid EMBL-3, was screened forclones having homology to cDNA clone TA39. Standard hybridizationmethods were used, such as are described in Sambrook et al., 1989.Candidate clones were purified by three or more cycles of pickingplaques, replating, and reprobing with a TA39 cDNA insert, untilconsistently hybridizing plaques were either purified or shown not bepresent.

Two distinguishable families of genomic tobacco DNA clones related tothe TA39 cDNA clone were identified, each represented by two overlappingclones within each family. One clone of each family was selected fordetailed characterization, designated clones 8B3 and 14B1. The region ofhomology with TA39 in each of these genomic clones, as well as theregions immediately upstream and downstream of these regions ofhomology, were mapped by restriction enzyme cleavage analysis and DNAhybridization.

These coding sequences and associated 5′ presumptive regulatory regionswere isolated as subclones and then further subcloned for sequencing.Thus, nested sets of deletions of each genomic clone were produced byusing exoIII and mung bean nucleases supplied in a kit by Stratagene.The nested deletions were sequenced by the dideoxy chain terminationmethod of Sanger with an automated DNA sequencer (Applied Biosystems373A) at the Nucleic Acids Facility of the Iowa State University. ThecDNA insert of TA39 was also sequenced for comparison. Within the regionof homology with the TA39 cDNA of a microspore-specific mRNA, genomicclone 8B3 is completely homologous with TA39, while the comparableportion of genomic clone 14B1 is about 90% homologous with TA39.

The starting points for transcription of the 14B1 and 8B3 genomic cloneswere mapped by primer extension experiments to a single nucleotide, 83bases upstream of the putative translational start site. A perfect TATAbox appears 31 bp upstream of the mapped start of transcription in eachclone, and a major open reading frame of 110 amino acids is intactdownstream of the start of transcription in both clones (i.e., at theposition designated “+83” relative to the transcription initiationsite). Both clones also have a polyadenylation recognition site, 29 bpand 37 bp downstream of a translational stop codon in clones 14B1 and8B3, respectively.

Transformation Methods. Transformation methods for dicots include anumber of different well-known methods for direct DNA delivery.Preferred is particle biolistics bombardment of leaf explants. Othermethods include Agrobacterium delivery to explants; Agrobacteriumcocultivation of protoplasts; electroporation; PEG uptake or otherdirect DNA delivery into protoplasts and the like. A preferred methodfor monocots such as corn is delivery of DNA to the treated cells bybombardment, but other methods such as electroporation can also be used.

Cells of a plant are transformed with the foreign DNA sequence of thisinvention in a conventional manner. If the plant to be transformed issusceptible to Agrobacterium infections, it is preferred to use a vectorcontaining the foreign DNA sequence, which is a disarmed Ti-plasmid. Thetransformation can be carried out using procedures described, forexample, in EP 0 116 718 and EP 0 270 822. Preferred Ti-plasmid vectorscontain the foreign DNA sequence between the border sequences, or atleast located upstream of the right border sequence. Other types ofvectors can be used for transforming the plant cell, using proceduressuch as direct gene transfer (see, for instance, EP 0 237 356, PCTpublication WO/85/01856 and EP 0 275 069); in vitro protoplasttransformation as described, for example, in U.S. Pat. No. 4,684,611;plant virus-mediated transformation as taught in EP 0 067 553 and U.S.Pat. No. 4,407,956, for example; and liposome-mediated transformation asdescribed in U.S. Pat. No. 4,536,475, among others.

If the plant to be transformed is corn, recently developedtransformation methods are suitable such as the methods described forcertain lines of corn by Fromm et al., 1990, and Gordon-Kamm et al.,1990.

If the plant to be transformed is rice, recently developedtransformation methods can be used such as the methods described forcertain lines of rice by Shimamoto et al., 1990, Datta et al., 1990,Christou et al., 1991, and Lee et al., 1991.

If the plant to be transformed is wheat, a method analogous to thosedescribed above for corn or rice can be used. Preferably for thetransformation of a monocotyledonous plant, particularly a cereal suchas rice, corn or wheat, a method of direct DNA transfer, such as amethod of biolistic transformation or electroporation, is used. Whenusing such a direct transfer method, it is preferred to minimize the DNAthat is transferred so that essentially only the DNA sequence of thisinvention, the QM maize gene and associated regulatory regions, isintegrated into the plant genome. In this regard, when a DNA sequence ofthis invention is constructed and multiplied in a plasmid in a bacterialhost organism, it is preferred that, prior to transformation of a plantwith the DNA sequence, plasmid sequences that are required forpropagation in the bacterial host organism, such as on origin ofreplication, an antibiotic resistance gene for selection of the hostorganism, and the like, be separated from the parts of the plasmid thatcontain the foreign DNA sequence.

TUNGSTEN/DNA PROTOCOL FOR DUPONT HELIUM GUN (PARTICLE BIOLISTICBOMBARDMENT METHOD OF TRANSFORMATION)

Weigh 60 mg 1.8 μm tungsten: put into 15 ml centrifuge tube

Add 2 ml 0.1M HnO₃: Sonicate on ice for 20 minutes

Withdraw HNO₃: Add 1 ml sterile deionized water and transfer sample to a2 ml Sarstedt tube. Sonicate briefly

Centrifuge to pellet particles

Withdraw H₂O: Add 1 ml 100% EtOH-Sonicate briefly

Centrifuge to pellet particles

Withdraw H₂O: Add 1 ml 100% EtOH-Sonicate briefly

Centrifuge to pellet particles

Withdraw EtOH. Add 1 ml sterile deionized water.

Sonicate.

Pipet 250 μl of suspension into 4, 2 ml tubes.

Add 750 μl of sterile deionized H₂O to each tube.

Freeze tungsten sample between use.

Pipet 50 μl tungsten/H₂O suspension into 1.5 ml tube

(Sonicate first)

Add 10 μg DNA, Mix

Add 50 μl 2.5M CaCl₂. Mix

Add 20 μl 0.1M Spermidine. Mix

Sonicate briefly. Centrifuge for 10 seconds at 10,000 RPM.

Withdraw supernatant. Add 250 μl 100% EtOH. Sonicate briefly.

Centrifuge at 10,000 RPM for 10 seconds

Withdraw supernatant. Add 60 μl 100% EtOH.

Transformation of maize:

Friable embryogenic Type II callus (Armstrong, 1991) was initiated from1-2 mm zygotic embryos isolated from A188 plants pollinated with B73,and maintained as described in Register et al., 1994. Callus wascultured biweekly for 4-6 months prior to transformation. Fortransformation, the callus was suspended in liquid culture medium andsieved through a 710 μm filter mesh, resuspended at a density of 40mg/ml. 200 mg callus cells were distributed evenly on a glassf iberfilter and used for particle bombardment as described in Register etal., 1994, except that 1.0 μm tungsten particles were used in place ofgold. Transformant selection and plant regeneration was performed asdescribed in Register, et al.; however, the concentration of bialophoswas elevated to 3 mg/L in all appropriate culture media.

Protocol Por Corn Transformation to Recover Stable Transgenic Plants

Day - 1 Cells are placed in liquid media and sieved (710 um). 100-200 mgof cells are collected on 5.5 cm glass fiber filter over an area of 3.5cm. Cells are transferred to media and incubated overnight.

Day - 8 Filter and cells are removed from media, dried and bombarded.Filter and cells are placed back on media.

Day - 5 Cells on the filter are transferred to selection media (3 mgbialophos).

Day - 12 Cells on the filter are transferred to fresh selection media.

Day - 19 Cells are scraped from the filter and dispersed in 5 ml ofselection media containing 8.6% low melting point sea agarose. Cells andmedia ate spread over the surface of two 100 mm×15 mm plates containing20 ml of gel-rite solidified media.

Day - 40 Putative transformants are picked from plate.

Day - 61 Plates are checked for new colonies.

CITED DOCUMENTS

An, G., Mitra, A., Choi, H. K., Costa, M. A., An, K., Thornburg, R. W.,and Ryan, C.A. (1989). Functional analysis of the 3′ control region ofthe potato wound-inducible proteinase inhibitor II gene. Plant Cell1:115-122.

Armstrong, C. L., Green C. E., and Phillips, R. L., (1991). Developmentand availability of germplasm with high type II culture formationresponse. Maize Genetics Cooperative Newsletter. 65:92.

Bellomy, G. and Record, M. Jr. (1989) Biotechniques 7:1.

Brooks, J. E., Blumenthal, R. M., and Gingeras, T. R., (1993). Theisolation and characterization of the Escherichia coli DNA adeninemethylase (DAM) gene. Nucl Acids Res. 11:837-851.

Bowen, B. (1992). Anthocyanin genes as visual markers in transformedmaize tissues. In GUS Protocols: Using the GUS Gene as a Reporter ofPlant Gene Expression, S. R. Gallagher, ed. (New York: Academic Press,Inc.), pp. 163-177.

Brent, R. and Ptashne, M. (1985) A eukaryotic transcriptional activatorbearing the DNA specificity of a prokaryotic repressor, Cell 43;729-736.

Chen, J. J., Pal, J. K., Petryshyn, R., Kuo, I., Yang, J. M., Throop, M.S., Gehrke, L. and London, I. M. (1991). Eukaryotic translationinitiation kinases. PNAs 88, 315-319.

Colasanti, J., Tyers, M. and Sundaresan, V., 1991. Isolation andCharacterization of cDNA clones encoding a functional P34 cdc2 homologuefrom Zca mags PNAs 88, 3377-3381.

Czako, M. and An, G. (1991) Expression of DNA coding for Diptheria toxinChain A is toxic to plant cells. Plant Physiol. 95 687-692.

Dennis, E., Gerlach W., Pryor, A., Bennetzen, J., Inglis, A., Llewellyn,D., Sachs, M., Ferl, R., and Peacock, W. (1994). Molecularcharacterization of the maize Adhl gene. Nucl. Acids Res. 12:3983-3990.

DeWet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani,S. (1987). Firefly luciferase gene: Structure and expression inmammalian cells. Mol. Cell. Biol. 7:25-737.

Droge, W., Broer, I., and Puhler, A. (1992) Transgenic plants containingthe phoshinothricin-N-acetyltransferase gene metabolize the herbicideL-phosphinothricin (glufosinate) differently from untransformed plants.Planta 187:142-151.

Farmer, A. A., Loftus, T. M., Mills, A. A., Sato, K. V., Neill, J.,Yang, M., Tron, T., Trumpower, B. L. and Stanbridge, E. G. (1994) Hum.Mol. Genet. 3, 723-728.

Fromm et al. (1990) Bio/Technology 8:833.

Gallie, D. R., Sleat, D. E., Watts J. W., Turner P. C., and Wilson, T.M. A. (1987). The 5′-leader sequence of tobacco mosaic virus RNAenhances the expression of foreign gene transcripts in vitro and invivo. Nucl. Acids Res. 15:3257-3273.

Gardner, R. C., Howarth, A. J., Hahn, P., Brow-Luedi, M., Shepherd R.J., and Messing, J. C. (1981). The complete nucleotide sequence of aninfectious clone of cauliflower mosaic virus by M13mp7 shotgunsequencing. Nucl. Acids Res. 9:2871-2888.

Goff, S. A., Cone, K. C., and Fromm, M. E., (1991). Identification offunctional domains in the maize transcriptional activator C1: Comparisonof wild-type and dominant inhibitor proteins. Genes Dev. 5,289-309.

Goldberg, R. B., Beals, T. P. and Sanders, P. M., (1993). Antherdevelopment: basic principles and practical applications. Plant Cell5:1217-1229.

Golemis, E. A., and Brent, R. (1992). Fused protein domains inhibits DNAbinding by LexA. Mol. & Cell Biol. 12:3006-3014.

Gordon-Kamm et al. (1990) Transformation of maize cells and regenerationof fertile transgenic plants, The Plant Cell 2:603-618.

Herskowitz, J. (1987). Functional inactivation of genes by dominantnegative mutations, Nature 329:219-222.

Invitrogen, Subtractor™ I Subtraction Kit for cDNA Probe Generation,Instruction Manual, version 2.3.

Koltunow et al. (1990) “Different temporal and spatial gene expressionpatterns occur during anther development.” Plant Cell 2:1201-1224.

Register, J. C., Peterson, D. J., Bell, P. J., Bullock, W. P., Evans, I.J., Frame, B., Greenland, A. J., Higgs, N. S., Jepson, I., Jiao, S.,Lewnau, C. J., Sillick, J. M., and Wilson, H. M. (1994). Structure andfunction of selectable and non-selectable transgenes in maize afterintroduction by particle bombardment. Plant Mol. Biol. 25:951-961.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). MolecularCloning, A Laboratory Manual. Cold Spring Harbor Press.

Shimamoto et al. (1990) Fertile transgenic rice plants regenerated fromtransformed protoplasts, Nature 338:274.

Su, T. Z., and El-Gewely, M. R., (1988). A multisite-directedmutagenesis procedure using T7 DNA polymerase: Application forreconstructing a mammalian gene. Gene 69:81-89.

Thompson, C. J., Movva, N. R., Tizard, R., Crameri R., Davies, J. E.,Lauwereys, M., and Botterman, J. (1987). Characterization of theherbicide-resistance resistance gene bar from Streptomyceshygroscopicus. EMBO J. 6:2519-2523.

EP 0 116 718

EP 0 270 822

EP 0 237 356

EP 0 275 069

EP 0 067 553

WO/85/01856

U.S. Pat. No. 4,684,611

U.S. Pat. No. 4,407,956

U.S. Pat. No. 4,536,475

What is claimed is:
 1. A recombinant DNA molecule comprising: (i) a DNAsequence encoding a gene product which when expressed in a plantinhibits or disrupts pollen formation or function; (ii) an operatorwhich controls the expression of said DNA sequence by binding arepressor protein. operatively linked to said DNA sequence encoding agene product; and (iii) a promoter specific to cells critical to pollenformation or function operatively linked to said DNA sequence encoding agene product.
 2. The recombinant DNA molecule of claim 1 furthercomprising a selectable marker gene.
 3. The recombinant DNA molecule ofclaim 1, wherein said gene product is a cytotoxin.
 4. The recombinantDNA molecule of claim 3, wherein said cytotoxin is a diphtheria toxinA-chain.
 5. The recombinant DNA molecule of claim 1, wherein said DNAsequence encodes a cell cycle division mutant gene.
 6. The recombinantDNA molecule of claim 5, wherein said cell division mutant gene isselected from the group consisting of CDC gene from maize, WT gene andP68.
 7. The recombinant DNA molecule of claim 1, wherein said geneproduct is a methylase.
 8. The recombinant DNA molecule of claim 1,wherein said gene product is a DAM methylase.
 9. The recombinant DNAmolecule of claim 1, wherein said promoter is an anther-specificpromoter.
 10. A plant cell comprising the recombinant DNA molecule ofclaim
 1. 11. A plant comprising the recombinant DNA molecule of claim 1.12. A method of producing a hybrid plant from a reversibly male sterileplant comprising: (a) providing a first plant comprising a firstrecombinant DNA molecule comprising (i) a first DNA sequence encoding agene product which when expressed in a plant inhibits or disrupts pollenformation or function, (ii) an operator which controls the expression ofsaid first DNA sequence by binding a repressor protein operativelylinked to said first DNA sequence encoding a gene product, and (iii) afirst promoter specific to cells critical to pollen formation orfunction operatively linked to said first DNA sequence; (b) providing asecond plant which is male fertile, said second plant comprising asecond recombinant DNA molecule comprising (i) a second DNA sequenceencoding a DNA binding protein which is a repressor protein, and (ii) asecond promoter which controls expression of said second DNA sequence,wherein said DNA-binding protein can bind to the operator of said firstrecombinant DNA molecule to repress expression of said first DNAsequence; and (c) crossing said first plant with said second plant toform a hybrid plant which is male fertile.
 13. A method of producinghybrid seed from a reversibly male sterile plant according to claim 12further comprising: (d) harvesting hybrid seed with restored fertilityfrom said crossing of step (c).